Solvent-Cage-Controlled Radical Flux and Reactive Adduct Intermediates Govern Azo-Initiated Oxidation

Abstract

Abstract Radical-mediated oxidation underlies processes ranging from oxidative stress in biological systems to antioxidant evaluation in food and pharmaceutical chemistry. In widely used azo-initiated systems such as those based on 2,2′-azobis (2-amidinopropane) (AAPH), radical flux is typically assumed to directly follow initiator decomposition, and antioxidant activity is interpreted as a single-step radical-scavenging process. However, the extent to which solvent-cage recombination and reactive intermediates modulate the effective radical flux and overall reaction kinetics remains unresolved. Here, we combine quantitative solution NMR spectroscopy with LC-MS to establish a kinetic and mechanistic framework for AAPH-induced oxidation of cinnamic acid derivatives. We show that classical single-step scavenging models fail to capture the experimentally observed concentration-dependent kinetics. Instead, oxidation proceeds through the formation of multiple covalent radical adducts that act as kinetically competent and reactive intermediates. Through complementary NMR, high-resolution mass spectrometry (HRMS), and LC-MS/MS analyses, we achieve direct structural characterization of these intermediates, revealing the formation of imidazolidine-type heterocycles arising from radical addition to the α,β-unsaturated side chain. By globally analyzing the time-dependent evolution of AAPH, its hydrolysis products, and radical recombination and disproportionation products, we obtained an experimentally constrained solvent-cage escape efficiency for AAPH-derived radicals (f ≈ 0.35), demonstrating that the effective radical flux is strongly decoupled from the overall rate of AAPH consumption. These results establish AAPH-mediated oxidation of cinnamic acid derivatives as a flux-controlled, multistep radical process governed by solvent-cage dynamics and intermediate reactivity. More broadly, our results redefine how radical flux and antioxidant activity are interpreted in azo-initiated systems, demonstrating that both are governed by hidden kinetic processes rather than simple scavenging. This study establishes a general NMR- and MS-based quantitative framework for probing radical reactivity in complex environments and challenges conventional interpretations of widely used antioxidant assays and radical-based screening platforms.